Bioactivities of β-mangostin and its new glycoside derivatives synthesized by enzymatic reactions

Beta-mangostin is a xanthone commonly found in the genus Garcinia. Unlike α-mangostin, to date, there have only been a few studies on the biological activity and derivatization of β-mangostin. In this study, two novel glycosylated derivatives of β-mangostin were successfully synthesized via a one-pot enzymatic reaction. These derivatives were characterized as β-mangostin 6-O-β-d-glucopyranoside and β-mangostin 6-O-β-d-2-deoxyglucopyranoside by TOF ESI/MS and 1H and 13C NMR analyses. Beta-mangostin showed cytotoxicity against KB, MCF7, A549 and HepG2 cancer cell lines, with IC50 values ranging from 15.42 to 21.13 µM. The acetylcholinesterase and α-glucosidase inhibitory activities of β-mangostin were determined with IC50 values of 2.17 and 27.61 µM, respectively. A strong anti-microbial activity of β-mangostin against Gram-positive strains (Bacillus subtilis, Lactobacillus fermentum and Staphylococcus aureus) was observed, with IC50 values of 0.16, 0.18 and 1.24 µg ml−1, respectively. Beta-mangostin showed weaker activity against Gram-negative strains (Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa) as well as Candida albicans fungus, with IC50 and MIC values greater than the tested concentration (greater than 32 µg ml−1). The new derivatives of β-mangostin showed weaker activities than those of β-mangostin, demonstrating the important role of the hydroxyl group at C-6 of β-mangostin in its bioactivity.


Materials
Beta-mangostin and α-mangostin were purchased from Chengdu Biopurify Phytochemicals, Ltd. Recombinant Escherichia coli (BL21 (DE3)) strains were kindly provided by Prof. Jae Kyung Sohng, Sun Moon University, Korea. The cell lines and microorganisms were derived from the American Type Culture Collection (ATCC). α-Glucosidase (CAS no. 900-42-7, Sigma) was from Saccharomyces cerevisiae. All chemicals were of analytical grade and available.
The reactions were kept at 37°C under shaking conditions (100 rpm) for 5 h and tested by thin-layer chromatography (ethyl acetate : methanol : water = 8 : 1.5 : 0.5). Depending on the yield of each system, β-mangostin substrate was added to achieve maximum conversion. The final products were analysed by high-performance liquid chromatography (HPLC).
The purified products were dried, lyophilized, dissolved in DMSO-d 6 and analysed using 1 H and 13 C NMR (nuclear magnetic resonance) spectroscopy (Bruker 600 MHz spectrometer) and ESI/MS (LC-MSD-Trap-SL).

Cytotoxicity assay
The cytotoxic activities against the cancer cell lines KB epidermal carcinoma, A549 lung cancer, HepG2 liver cancer and MCF-7 breast cancer, as well as the healthy cell line HEK293 embryonic kidney cells, were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [11,12]. Briefly, cell lines were grown into log phase at 37°C, 5% CO 2 in DMEM-Dulbecco's modified Eagle's medium and 10% fetal bovine serum. A concentration of 1 × 10 4 -3 × 10 4 cells ml −1 was prepared depending on the cell line used for the experiment by adding 10 µl of the sample and 190 µl of the cell suspension, followed by incubation under standard conditions. After 72 h of culture, 10 µl of MTT (5 mg ml −1 ) was added and incubated for 4 h. Dimethyl sulfoxide (100 µl) was added to dissolve formazan crystals and the density was measured at 540 nm using a BioTek spectrophotometer. The inhibition of cell growth was expressed as IC 50 (the half-maximal inhibitory concentration) value. Ellipticine was used as reference.

Acetylcholinesterase inhibitory activity assay
For the assay, acetylthiocholine iodide (ATCI) was used as a substrate for the reaction and 5,5 0 -dithiobis-(2nitrobenzoic acid) (DTNB) was used to measure AChE activity [13,14]. The reaction (200 µl) on a 96-well plate was performed by mixing Tris-Cl buffer (pH 8.0), tested sample in a twofold serial dilution, AChE (0.25 IU ml −1 ) and incubating at 25°C, 15 min before adding 2.4 mM DTNB and 2.4 mM ATCI. The reaction mixture was mixed well and further incubated for 15 min and was measured at 412 nm (A). The ability of the tested samples to inhibit AChE activity was determined as the IC 50 (μM), the concentration that reduced 50% of AChE activity with reference to the control (donepezil).

Alpha-glucosidase inhibitory assay
The α-glucosidase inhibitory activity assay [15,16] was conducted using a reaction mixture containing phosphate buffer (100 mM; pH 6.8), p-nitrophenyl-α-D-glucopyranoside ( pNPG) (2.5 mM) and samples at various concentrations. The reactions were initiated by adding α-glucosidase (0.2 U ml −1 ), incubated at 37°C for 30 min and quenched by adding Na 2 CO 3 (50 mM). The absorbance of the reaction was determined using a BioTek instrument at a wavelength of 410 nm (A). The inhibition of α-glucosidase activity was determined as IC 50 (μM) with acarbose was a positive control.

Anti-microbial activity assay
Bacterial and fungal strains (Bacillus subtilis, Staphylococcus aureus, Lactobacillus fermentum, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica and Candida albicans) were activated on TSA (Tryptic Soy Agar) and SA (Sabouraud-4% dextrose agar) media, respectively. Before the experiment, the microbial strains were grown in the corresponding liquid media at a concentration of approximately 5 × 10 5 CFU ml −1 for bacteria and 1 × 10 3 CFU ml −1 for fungi. The microbial suspension (190 µl) was mixed with 10 µl of the test sample, which was serially diluted twofold, and the microplate was incubated at 37°C for 16-24 h. The MIC value was determined for the well with the lowest sample concentration that completely inhibited microbial growth [17,18]. Compounds (1) and (2) were purified by preparative HPLC and analysed by mass spectrometry and NMR. The TOF ESI/MS of compound (1) showed a peak of [M + H] + ions at m/z 571.2457, which confirmed the glycoside product of β-mangostin conjugated with the 2-deoxyglucose residue. Similarly, the ion peak [M + H] + of compound (2) was at 587.0, corresponding to the formula C 31 H 38 O 11 , a β-mangostin glucoside. The structures of these new products were further analysed using 1 H and 13 C NMR spectroscopy.
The cytotoxic activity of β-mangostin has also been reported in other cell lines, and its IC 50 value varies widely. In HeLa cervical cancer cells, the IC 50 value was 27.2 µM [23]. On glioma cells, β-mangostin inhibited cell survival at IC 50 values of 4.8, 5.8 and 10.3 µM for C6, U251 and T89G cells, respectively [24]. Several mechanisms of β-mangostin activity have also been revealed, such as inhibitory effects on DNA polymerases and topoisomerases, cell cycle arrest at the G1 and S phases, apoptosis in C6 cells, and mitochondrial function impairment and suppression of the PI3K, AKT and mTOR signalling pathways [24].
Alpha-mangostin and β-mangostin exhibited different toxicities in the five tested cell lines. Betamangostin exhibited a fairly stable effect with IC 50 values ranging from 15.42 to 21.13 μM, while αmangostin exhibited IC 50 values with broader range, from 0.24 μM to 81.9 μM depending on the cell line (table 2). In terms of molecular structure, these molecules differ only at the C-3 position, with C3-OH in α-mangostin and C3-OCH 3 in β-mangostin, suggesting that C3-OH may interact with different signalling molecules in different cell lines. In addition, the C6-glycoside (2-deoxyglucose) of α-mangostin had similar toxicity to α-mangostin [26], whereas the 2-deoxyglucoside of β-mangostin showed 4.8-fold lower activity than that of alycone. Therefore, further studies on the mechanism of these derivatives need to be conducted to better evaluate the relationship between their structure and cytotoxic activity to develop anti-cancer drugs.

Inhibition of acetylcholinesterase activity
AChE plays the main role in hydrolysis of acetylcholine (90%) along with butyrylcholinesterase (BuChE) to regulate cholinergic neurotransmission [27][28][29]. AChE inhibitors can delay the progress of mental illness and reduce neuropsychiatric symptoms, thus providing a rational therapeutic approach to Alzheimer's disease treatment. The results in table 3 show that β-mangostin had a significant inhibitory effect on AChE, whereas glycoside derivatives (1) and (2) both had weaker effects. The AChE inhibitory activity of β-mangostin was similar to that of α-mangostin (2.14 μM), as reported by Khau et al. [30].
Currently, four drugs are used for treatment of Alzheimer's disease: donepezil, rivastigmine, galantamine and the glutamate antagonist memantine. Galantamine, an alkaloid extracted from the Amaryllidaceae family, is a naturally occurring AChE inhibitor. The advantage of natural AChE inhibitor substances is that in addition to anti-AChE activity, there are other beneficial activities, such as antioxidant activity [33][34][35]. Several recent studies have been conducted to identify and isolate natural molecules applicable to the design and development of new anti-Alzheimer's disease drugs [36,37]. Our study also found that the natural compound β-mangostin has high potential for AChE inhibition (IC 50 value of 2.17 µM) in comparison to synthesized derivatives. Similar natural xanthone compounds including α-mangostin and γ-mangostin had strong inhibitory activity against AChE with IC 50 values of 2.14 µM and 1.31 µM, respectively [30]. In addition to the causes of Alzheimer's disease by impaired neurotransmission, some others have also been studied and become the target in the treatment of Alzheimer's disease such as: accumulation of pathological proteins, e.g. amyloid-β protein, tau tangles and neuroinflammation activation, and increased oxidative stress [38][39][40][41].

Inhibition of α-glucosidase activity
Alpha-glucosidase is an important enzyme that affects the blood glucose levels. These enzyme inhibitors slow the release of D-glucose and reduced blood sugar levels. Therefore, one strategy for the treatment of diabetes is inhibition of α-glucosidase.
From the results in table 4, β-mangostin showed strong inhibitory activity against α-glucosidase (IC 50 = 27.62 µM) when compared with the positive control acarbose (IC 50 = 208.62 µM), with an inhibitory potency ratio of 7.55. All glycoside derivatives seemed to lose this activity, with IC 50 values higher than the tested concentrations (greater than 124 µM for β-Man2DG and greater than 50 µM for β-ManGlc). This result is similar to that of α-mangostin, with α-glucosidase inhibitory activity at an IC 50 of 31.1 µM, while synthetic compounds (CS1-CS4, derivatives of xanthenone substituted at position 1 with various side chains) lost this activity [42].
The structural relationship and α-glucosidase inhibitory activity were also determined by analysing the IC 50 values of the investigated substances, such as synthetic hydroxylxanthones [43]. The results showed that all eight hydroxylated derivatives of xanthone had 21 times higher α-glucosidase inhibitory activity than that of the parent xanthone, and there was a correlation between enzyme inhibitory activity and the number of hydroxyl groups, such as 1,3,7-trihydroxyxanthone, which had 12-fold stronger activity than 1-hydroxyxanthone [43]. Similarly, the α-glucosidase inhibitory activities of α-mangostin, β-mangostin and γ-mangostin were investigated in a study by Ryu et al. [44], showing a close relationship between the number of hydroxyl groups in the A and B rings and inhibitory potential in the order γ-mangostin (IC 50 = 1.5 μM) > α-mangostin (IC 50 = 5.0 μM) > β-mangostin  [44]. This may also explain why glycosylated derivatives of β-mangostin had lower α-glucosidase inhibitory activity when the C6-OH group was glycosylated.

Anti-microbial activity
The results in table 5 show that β-mangostin was able to inhibit B. subtilis and L. fermentum with the same MIC values of 0.5 μg ml −1 and IC 50 values of 0.16 μg ml −1 and 0.18 μg ml −1 , respectively. For the remaining strains, β-mangostin and its derivatives (1) and (2) exhibited weak inhibitory activity, with MIC values higher than the tested concentrations. In a previous report on the antibacterial activity of xanthone against S. aureus (MRSA; methicillinresistant strain) and Enterococcus (VRE; vancomycin-resistant strain), β-mangostin showed no antibacterial activity (MIC > 200 μg ml −1 ), whereas αand γ-mangostin exhibited the highest activities among the eight xanthones studied (MICs ranging from 3.13 to 6.25 μg ml −1 ) [45]. Based on those results, it was suggested that the C3-OH, C6-OH and C2-prenyl chain were essential for antibacterial activities of α-mangostin and γ-mangostin [45]. Therefore, further research on the antibacterial mechanism of xanthones and their derivatives is required to understand the role of these functional groups.

Conclusion
The natural compound β-mangostin has potential activities associated with cancer cell toxicity, inhibition of AChE and α-glucosidase, and Gram-positive antibacterial activity. Two new derivatives of β-mangostin, β-mangostin 6-O-β-D-2-deoxyglucopyranoside (1) and β-mangostin 6-O-β-D-glucopyranoside (2), were  synthesized by enzymatic methods. Although the biological activities of these derivatives have been reduced compared to those of β-mangostin, further research is required to clarify the role of the functional groups of these derivatives in the mechanisms of anti-cancer, antibacterial, antidiabetic and anti-Alzheimer effects.
Ethics. The human cell lines that we used in this study were obtained from commercial sources (ATCC) and we did not require ethical approval.
Data accessibility. The data are provided in electronic supplementary material [46]. Declaration of AI use. We have not used AI-assisted technologies in creating this article. Authors' contributions. T.T.L.: conceptualization, project administration, writing-original draft, writing-review and